Targeted treatment of injured nestmates with antimicrobial compounds in an ant society

Infected wounds pose a major mortality risk in animals. Injuries are common in the ant Megaponera analis, which raids pugnacious prey. Here we show that M. analis can determine when wounds are infected and treat them accordingly. By applying a variety of antimicrobial compounds and proteins secreted from the metapleural gland to infected wounds, workers reduce the mortality of infected individuals by 90%. Chemical analyses showed that wound infection is associated with specific changes in the cuticular hydrocarbon profile, thereby likely allowing nestmates to diagnose the infection state of injured individuals and apply the appropriate antimicrobial treatment. This study demonstrates that M. analis ant societies use antimicrobial compounds produced in the metapleural glands to treat infected wounds and reduce nestmate mortality.

in isolation exposed to different potential pathogens cultured from the soil (Burkholderia, Rhizopus, Pseudomonas; OD=0.1) or surface soil pathogens (Supplementary Fig. 3).(c) Statistical results for infected and sterile ants (P.aeruginosa: OD=0.05) in isolation and with nestmates in the nest (Fig. 2a).(d) Statistical results of infected ants (P.aeruginosa: OD=0.05) kept in isolation or inside sub-colonies containing workers with or without plugged MG openings (Fig. 4; Supplementary Fig. 5).

Supplementary Fig. 1 |
Fig. 1A.Multiple bars of the same color indicate different amplicon-sequence variants (ASVs) belonging to the same genera.ADONIS: Treatment: F(1,39)=17.45;R 2 =0.31;P<0.001.N=10 for all groups.(c) Microbial culture of surface soil grown on an agar plate (mostly showing fungal mycelia of Rhizopus with black spores).(d) Bacterial cultures of isolated Pseudomonas aeruginosa strains from the soil.Cultures in panels c and d were repeated 10 times showing the same result.
Cox proportional hazard models for survival of infections.The reference were sterile ants in isolation with colony of origin as random factor.(a) Statistical results for infected ants (surface soil pathogens: OD=0.1) in isolation and inside the nest (Fig. 1c).(b) Statistical results for infected ants kept

Rhizopus Supplementary Fig. 4 | Differences in wound care duration with and without metapleural gland secretions.
Duration of wound care with and without MG secretions for the first 24 hours after injury d Supplementary Fig. 3 | Survival probability for different pathogen types.Kaplan -Meier cumulative survival rates of workers kept in isolation chambers.All individuals had one hind leg cut off at the femur, the wound was then exposed to a sterile PBS solution (green, sterile, n=104), a mix of either surface soil pathogens (red, Soil OD=0.5, n=20) or isolated pathogens diluted in PBS (OD=0.1):P.aeruginosa(blue, Pseudomonas, n=61), Burkholderia (brown, n=16), Rhizopus (pink, n=16), or Rhizopus with Burkholderia (dark blue, n=16).The treated ants were observed for 36 hours and the time of death noted.Statistical significance tested with a mixed-effects Cox proportional hazards regression model (Supplementary Fig.RhizopusFig.S1 Different concentrations of soil and Pse in isolation.

Supplementary Fig. 5 | Importance of MG secretions on survival of sterile and infected ants. Kaplan
colonies (darker color shade, MG open).n=12pertreatment.Detailed statistical results in Supplementary Fig.2dand Supplementary Table 5, significant differences were calculated using a twosided least square means with Holm-Bonferroni correction.

Supplementary Table 2 | Statistical differences between infected and sterile ants in pathogen abundance for the different bacterial genera at 2 and 11 hours presented in Fig. 1b.
Supplementary Table 3 |Statistical differences for mortality between the different treatments presented in Fig 1c.Mixed effects Cox proportional hazards regression model: Random factor: Colony: Variance: 0.11, Std.Dev.0.33.Likelihood ratio test of model vs intercept only model: X 2 3= 16.4,P<0.001.Post-hoc analysis with least square means with Holm-Bonferroni correction for the 4 groups.

Supplementary Table 4 | Statistical differences for mortality between the different treatments presented in Fig 2a
. Mixed effects Cox proportional hazards regression model: Random factor: Colony: Variance: 0.45, Std.Dev.0.66.Likelihood ratio test of model vs intercept only model: X 2 3= 24.59, P<0.001.Post-hoc analysis with least square means with Holm-Bonferroni correction for the 4 groups.

Table 6 | Statistical differences for mortality between the different treatments presented in Supplementary Fig. 5. Mixed
acrylic color both in the focal ant and nestmates; open: MG was kept open both in the focal ant and nestmates; Sterile: wound of focal ant was exposed to PBS; Infected: wound of focal ant was exposed to 0.05 OD of P. aeruginosa.

Table 10 | List of genes putatively implicated in CHC production and lipid metabolism being differentially expressed between sterile and infected ants presented in Fig. 5. Positive Log2FoldChange
values correspond to genes up-regulated in infected ants.Significant differences were calculated using a two-sided Wald test and corrected for multiple testing using the Benjamini and Hochberg

Table 11 | Immune system related genes differentially expressed between sterile and infected ants presented in Fig. 5.
Positive Log2FoldChange values correspond to genes up-regulated in infected ants.Significant differences were calculated using a two-sided Wald test and corrected for multiple testing using the Benjamini and Hochberg method